Ultramicrotome device

ABSTRACT

In a process for cutting sections from a probe for microscopic analysis, an ultramicrotome device is used having a blade with a cutting edge, the cutting edge extending at least approximately in a first direction. The process includes the steps of: vibrating said blade in the first direction; and moving the blade relative to the probe to be cut in a second direction, the second direction being perpendicular to the first direction. This eliminates, or at least strongly reduces, compression of the cut sections.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/718,636, filed Nov. 22, 2000, which is herebyincorporated by reference in its entirety and which is acontinuation-in-part of U.S. patent application Ser. No. 09/207,284,filed Dec. 8, 1998.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a process for cutting sectionsfrom a probe for microscopic analysis by using an ultramicrotome device.

[0004] 2. Description of Related Art

[0005] Microtomes and ultramicrotomes are used to cut thin respectiveultra-thin sections from a sample for microscopic analyses. The sampleis mounted on a cross-slide which can be advanced horizontally in stepsaccording to the desired thickness of the sections and vertically forperforming the cutting operation. A cutting blade with a horizontalcutting edge is mounted on a holder. Microtomy is concerned with a rangeof thickness of 0.5 to 50 μm of the sections and is mainly used foroptical microscopy. Ultramicrotomy is concerned with a range ofthickness of 10 to 100 nm of the sections. This range of thickness isrequired for transmission electron microscopy. Ultramicrotomy has provedto be a very fast and efficient technique not only for TEM but also forsurfacing samples for STM and AFM.

[0006] In microtomy mainly steel blades are used for cutting. GermanPatent No. 913 112 discloses an older type of a microtome in which thecutting blade is horizontal and the sample advances upwardly in stepsbetween the cuts. The blade is fastened between two parallel leafsprings and driven by magnets for oscillating movement parallel to thecutting edge. The cutting edge of a steel blade is relatively rough whenviewed under an electron microscope and relatively blunt. With theoscillating motion of the blade, therefore, a sawing action is achieved:the jags of the cutting edge act like saw teeth.

[0007] This sawing action of the blade in a microtome is also describedin the DDR Patent No. 146 199, in which the blade is driven by anelectroacoustical transducer at high frequency, and in the BelgianPatent No. 440 928 which uses an ultrasound emitter to oscillate theblade.

[0008] In ultramicrotomy the sections are so thin that extreme care mustbe taken to shield the ultramicrotome from all possible external andinternal vibrations because they would adversely affect the cuttingresult. It therefore seemed impossible to transfer the sawing action ofthe cutting blade known from microtomy to an ultramicrotome. For thisreason much sharper and perfectly rectilinear cutting edges are requiredin ultramicrotomy. This has been achieved by cutting blades of diamond.U.S. Pat. No. 4,697,489 describes a holder with such a diamond cuttingblade for ultramicrotomes. With the perfectly rectilinear cutting edge,even when viewed under an electron microscope, of a diamond blade nosawing action can be achieved as with steel blades.

[0009] For the ultramicrotomy at room temperature, usually the cuttingis performed on a knife mounted in a boat which contains water. Thewater forms a horizontal surface behind the cutting edge of the knife.Due to the surface tension the sections float on the water surface andcan be collected. The water acts as a lubricant during sectioningprocess.

[0010] However, in ultramicrotomy a different problem arises which doesnot occur in microtomy: the problem of section compression. Thisphenomenon occurs at a thickness of the sections below 100 nm. Dependingon the mechanical properties of the sample (flexibility) and on thesectioning angle φ of the knife the sections undergo considerabledistortion (compression) during cutting (FIG. 1). In FIG. 1, 1designates the diamond blade or knife with the cutting edge 2. 3 is thesample. The sample 3 may be one of a great variety of industrial orbiological samples. A is the vertical movement of the sample 3. 4 is thecut section floating on a waterbed 5. 6 designates the direction ofcompression in the section 4. 7 is a region of intense shearing, and 8is the region of compression in the sample 3.

[0011] Water sensitive samples 3 have to be cut dry. Due to the missinglubrication and to the friction on the knife surface the sections 4 areeven more compressed as the ones cut on water. In cryo-UM most sampleshave to be cut dry. The amount of compression depends on differentfactors:

[0012] The sectioning angle of the knife.

[0013] The hardness of the sample.

[0014] The triboelectrical properties of the sample.

[0015] The most critical factor is the sectioning angle φ. Thesectioning angle φ is the sum of the wedge angle β of the knife 1 andthe clearance angle δ. It was shown that reducing the wedge angle βresults in a reduction of compression. However, the wedge angle β maynot be reduced ad infinitum. We have found an angle of 30° to be alimit. A further reduction of the wedge angle results in a lower cuttingedge 2 quality and in a considerably shorter service time of the knife1. In cryo-UM the compression in sections was found almost equal withthe sectioning angle φ. Therefore, a knife 1 working with a sectioningangle φ of 40° (wedge angle β30°, clearance angle δ10°) would result ina compression in the sections 4 of approximately 40%.

SUMMARY OF THE INVENTION

[0016] In order to preserve the original ultrastructure and form ofmatter, it would be desirable to eliminate the distortion (compression)in the sections 4.

[0017] The problem to be solved with the present invention is to createa process used in an ultramicrotome which reduces or eliminates thecompression of the sections. This problem is solved by the presentinventive process for cutting sections from a probe for microscopicanalysis, by using an ultramicrotome device having a blade, especially adiamond blade, with a cutting edge, wherein this cutting edge extends ina non-vibrated position at least approximately in a first direction.This process comprises the steps of vibrating said blade in the firstdirection and moving the blade relative to the probe to be cut in asecond direction, the second direction being perpendicular to the firstdirection.

[0018] Briefly stated, the device used in such a process comprises aholder and a block attached to the holder by at least one spring. Adiamond blade is attached to the block. The cutting edge of the blade issubstantially horizontal in operation. A vibrator cooperates with theblock to vibrate it substantially parallel to the cutting edge.Preferably, the vibrator comprises a piezoelectric transducer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a schematic diagram showing the effective sectioningangle a when the blade or knife 1 is moved in the direction of the edge2 during cutting;

[0020]FIG. 2 is a side view of a first embodiment according to thepresent invention;

[0021]FIG. 3 is a front view, partially in section, of the firstembodiment of FIG. 2;

[0022]FIG. 4 is a side view of a second embodiment according to thepresent invention;

[0023]FIG. 5 is a front view, partially in section, of the secondembodiment of FIG. 4;

[0024]FIG. 6 is a side view of a third embodiment according to thepresent invention; and

[0025]FIG. 7 is a front view of the third embodiment of FIG. 6.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0026] In the present invention an oscillating movement of the blade orknife 1 parallel to the cutting edge 2 and perpendicular to the cuttingdirection A is used to eliminate or at least strongly reduce compressionof the sections 4. When the knife 1 moves in the direction of thecutting edge 2 while the probe 3 moves in the direction A, an effectivecutting direction B results which forms an acute angle γ with thecutting edge 2 (FIG. 1). If y is the vertical movement of the probe 3per time unit and z is the effective relative movement between knife 1and probe 3 in the same time unit, it can be seen from FIG. 1 that${{\tan \quad \alpha} = \frac{x}{z}};{{\sin \quad \gamma} = \frac{y}{z}};{{\tan \quad \varphi} = {\frac{x}{y}.}}$

[0027] It follows:${\sin \quad {\gamma \cdot \tan}\quad \varphi} = {{\frac{y}{z} \cdot \frac{x}{y}} = {\frac{x}{z} = {\tan \quad {\alpha.}}}}$

[0028] When the knife 1 vibrates, the effective sectioning angle αvaries (maximum effective sectioning angle α equal to φ, minimaleffective sectioning angle α close to 0°). The theoretical value ofcompression reduction is as follows: An assumed mean effectivesectioning angle α depends on the amplitude C (mm) and the frequency ν(Hz) of the vibration and on the cutting speed v (mm/sec). Only smalleffective sectioning angles α are considered. Under this assumption itcan be shown that

tan α=(v/C·ν) tan φ.

[0029] To give an example, the following parameters are assumed:

φ=45°; ν=0.1 mm/sec; C=1 μm; ν=1 kHz.

[0030] It follows

tan α=(0.1 mm/sec)/(0.001 mm·1000 Hz)·1=0.1

[0031] resulting in a mean effective sectioning angle α of about 5.7°.

[0032] The theoretical assumptions seem to be correct because on aprototype the oscillating knife has shown to significantly reduce thecompression of the sections 4.

[0033] In ultramicrotomy the persons skilled in the art have takenextreme care to shield the microtome from all possible external andinternal vibrations because they adversely affect the cutting result.The inventor has overcome this prejudice and could show that byvibrating the knife 1 substantially parallel to the cutting edge 2, noadverse effect of the vibration was observed.

[0034] A first embodiment of the invention is shown in FIGS. 2 and 3.The blade 1 is sintered in a bronze holder 16 or vacuum brazed in atungsten carbide holder. The holder 16 is mounted on an inclined face ofa recess 17 in a block 18. The block 18 is mounted to a holder 19 bymeans of a leaf spring 20. The plane of the leaf spring 20 issubstantially vertical and perpendicular to the cutting edge 2. Thespring 20 is mounted to the block 18 and the holder 19 by flat plates 21and screws 22. Alternatively, the spring 20 may be designed as anintegral part of the block 18 and the holder 19.

[0035] An arm 23 extends upward from the base 24 of the holder 19. Thearm 23 has a cylindrical horizontal boring 25 and a slot 26 on one side.The cylindrical housing 29 of a vibrator 30 with a piezoelectrictransducer 31 and an actuating rod 32 is held in the boring 25 by meansof a screw 33. The spherical face end 34 of the rod 32 is slightlypressed against a plane face 35 of the block 18. The axis 36 of thevibrator 30 is parallel to the cutting edge 2. The spring 20 may beslightly bent towards the vibrator 30 in the unloaded state before thevibrator 30 is mounted in position such that with the deflection of thespring 20 required for the preload force of the block 18 against the rod32 the spring 20 gets plain and vertical. The axis 36 passes through thecenter of gravity 40 of the block 18.

[0036] The vibrator 30 is connected to an oscillator 37 by means of acable 38. Two adjustment knobs 39 on the oscillator 37 allow theselection of the amplitude and frequency of the oscillation of thevibrator 30. Preferably, the frequency is selected in the ultrasoundrange above 15 kHz. The required amplitude is then only in the range of10∝1000 nm.

[0037]FIGS. 4 and 5 show a second embodiment. Similar parts aredesignated with the same reference numerals so that a detaileddescription of those parts is omitted. The embodiment of FIGS. 4 and 5has two parallel leaf springs 20 of equal active length L. The upper andlower ends of the active length L of the two springs 20 lay inhorizontal planes which are parallel to the cutting edge 2. Thisarrangement has the advantages that the cutting edge 2 moves moreparallel to itself than in the first embodiment. In the first embodimentit makes a minute pendulum motion, and that vibrations around a verticalaxis are strongly restricted.

[0038] In this embodiment the piezoelectric thickness transducer 31 isdirectly attached, e.g., bonded with one of its plane end faces 46 to avertical face 47 of the block 18. A counter mass 48 is fastened to theopposite end face 49 of the transducer. Instead of or in addition todirectly bonding the faces 46 and 49 to the block 18 and counter mass48, a pressing force by springs 64 may be used which may bear againstarms 65 attached to the holder 19. This variant is shown in dash-dottedlines in FIG. 4.

[0039] This arrangement of the vibrator 30 has the advantage thatconsiderably higher accelerations of the block 18 towards the countermass 48 are possible. This is particularly of advantage when higherfrequencies are used, e.g., in the ultrasound range because theaccelerations increase with the square of the frequency.

[0040] The embodiment of FIG. 5 is shown in the variant for dryultramicrotomy, e.g., without the water 5 in a trough behind the blade1. Instead, the upper, horizontal face 55 of the block 18 has adepression 56 which is filled with a plastic insert 57 with a planeupper surface 58, the plane of which intersecting the front face 59 ofthe blade 1 at an angle of 75° to 85°, preferably about 80°. Therefore,when the blade 1 is set at the recommended clearance angle of 10°, thesurface 58 is exactly horizontal which greatly facilitates observationof the cut sections 4 with a stereo microscope, e.g., for sectionpick-up since no refocusing is required when moving the microscopehorizontally. A material with good triboelectrical properties for theinsert 57 is an epoxy resin.

[0041] Instead of the piezoelectric transducer 31, other types oftransducers could be used, e.g., magnetic transducers. A suitabletransducer would be a moving coil transducer similar to the one used inmoving coil loudspeakers. The moving coil would be mounted to the block18 and connected to the oscillator 37. The (e.g., permanent) magnetsurrounding the coil and acting as counterweight could be elasticallysuspended (e.g., like the block 18 in FIG. 4) on the holder 19. The axisof the coil would be coincident with the axis 36.

[0042]FIGS. 6 and 7 show a third embodiment. Similar parts are againdesignated with the same reference numerals. In this embodiment, theholder 19, the block 18 and the leaf spring 20 are manufactured from asingle piece of metal. The spring 20 is a web connecting the holder 19and the block 18. The holder 19 is mounted on a base 72. In operation,the block 18 oscillates with an amplitude a_(o) and with a frequency inradians ω=2π·ν, wherein ν is the frequency in Hz, in a horizontal firstdirection x parallel to the cutting edge 2. The oscillating movement isa_(o) sin ωt and the oscillating speed v_(h) is a_(o) ω cos ωt.

[0043] A first slide 73 is slidably guided on first guide rails 74 ofthe base 72 which extend in a horizontal second direction yperpendicular to the first direction x. The movement of the slide 73 iscontrolled by a first actuator 75 for stepwise advance of the probe 3towards the cutting edge 2 between successive cuts. Second guide rails76 are mounted on the slide 73 and extend in the vertical direction zwhich is perpendicular to the first direction x and the second directiony. A second slide 77 is slidably guided in the rails 76. The movement ofthe second slide 77 is controlled by a second actuator 78 which controlsthe vertical cutting speed v_(c) of the probe 3 relative to the cuttingedge 2.

[0044] A base 79 of a chuck 80 is mounted to the slide 77 by means of asecond leaf spring 81. The base 79, spring 81 and slide 77 are againshown as manufactured from a single metal block. The plane of the spring81 is horizontal, i.e., parallel to the cutting edge 2 and perpendicularto the plane of the spring 20. The chuck 80 clamps the probe or sample3. A second vibrator 82 is mounted on the chuck 80. It consists of apiezoelectric transducer 83, which is bonded with one face end to thechuck 80, and a counter mass 84 which is bonded to the opposite face endof the transducer 83.

[0045] In operation, the chuck 80 and therewith the probe 3 is advancedvertically by the actuator 78 with a constant cutting speed v_(c) forcutting. A vertical oscillation by the vibrator 82 is superimposed onthe cutting speed v_(c) with an amplitude b_(o) and a frequency 2ω whichis twice the oscillating frequency of the vibrator 30. The oscillatingmovement is b_(o) cos (2ωt−π/2) and the oscillating speed v_(v) is −2b_(o) ω sin (2ωt−π/2). The total vertical speed v_(p) of the probe istherefore

v _(p) =v _(c) +v _(v) =v _(c)−2 b _(o) ω sin (2ωt−π/2)

[0046] The vertical amplitude b_(o) and the frequency ω are now chosensuch that

2b _(o) ω≧v _(c)

[0047] In this way the actual vertical cutting speed v_(p) of the probeis zero or negative when the horizontal speed v_(h) is zero, i.e., whenωt=π/2+n·π where n is an integer number.

[0048] In other words, the phase angle, the amplitude b_(o) of thevertical oscillation and the frequency ω are chosen such that the actualvertical speed v_(p) of the probe is zero or negative when thehorizontal movement of the knife 1 reaches its reversal points.

[0049] It is also possible to vibrate the probe in a horizontaldirection, i.e., at least approximately parallel to the cutting edge ofthe blade. Preferably, the probe and the blade are vibrated such thatwhen the blade 1 reaches its reversal points, the probe is still moving,preferably at maximum speed and vice versa. Preferably, the probe andthe blade are vibrated at the same frequency, but not in the same phase.

[0050] By vibrating the probe either in a vertical or a horizontaldirection, section compression can be completely avoided even in thesereversal points.

[0051] As an example for the vibration in a vertical direction: when thehorizontal frequency ω is 2π·16 kHz=10⁵s⁻¹ and the advance speed v_(c)=2mm·s⁻¹ then the vertical amplitude b_(o) would have to be at least 10nm. The horizontal amplitude a_(o) is again considerably less than 1 μm.In the above example, with the requirement that tan ∝≦0.1 the horizontalamplitude a_(o) of the knife 1 would have to be at least 200 nm(a_(o)ω≦20 mm/s). With lower cutting speeds v_(c), the amplitudes a_(o)and b_(o) can be reduced accordingly.

[0052] Of course, the relative movement between the knife 1 and theprobe 3 can be achieved in different ways than the one specificallyshown in FIG. 6., e.g., the slide 73 and/or the slide 77 could beassociated with the holder 19 instead of with the chuck 80, or thehorizontal and vertical vibrations could be reversed, i.e., that theknife 1 oscillates vertically and the chuck 80 horizontally, or bothvibrations could be imparted on the same elements, knife 1 or chuck 80.

The invention claimed is:
 1. A process for cutting sections from a probefor microscopic analysis, by using an ultramicrotome device having ablade with a cutting edge, the cutting edge in a non-vibrated positionextending at least approximately in a first direction, the processcomprising the steps of: vibrating the blade in the first direction; andmoving the blade relative to the probe to be cut in a second direction,the second direction being perpendicular to the first direction.
 2. Theprocess according to claim 1, wherein the probe is cut in sectionshaving a thickness of about 10 to about 100 nm.
 3. The process accordingto claim 1, wherein the blade is vibrated with a maximum amplitude ofthe vibration of the blade of about 1 μm.
 4. The process according toclaim 1, wherein force is applied to a block of the ultramicrotomedevice, the block holding the blade.
 5. The process according to claim1, wherein the probe is vibrated in a third direction perpendicular tothe first and the second direction.
 6. The process according to claim 5,wherein the blade is vibrated in a first frequency and the probe isvibrated in a second frequency, the second frequency being twice thefirst frequency.
 7. The process according to claim 1, wherein the probeis vibrated in a third direction at least approximately parallel to thefirst direction.
 8. The process according to claim 7, wherein the probeand the blade are vibrated, such that when the blade reaches itsreversal points, the probe is still moving and vice versa.
 9. Theprocess according to claim 8, wherein the probe and the blade arevibrated at the same frequency, but in a different phase.
 10. Theprocess according to claim 7, wherein the blade is moved relative to theprobe in the second direction with a substantially constant cuttingspeed over a distance larger than a cross-sectional dimension of theprobe in the third direction.
 11. The process according to claim 1,wherein in the third direction, an amplitude b_(o) of vibration is usedwith b _(o) ≧v _(c)/2ω, wherein ω is the frequency in radians of thefirst vibrator and v_(c) is the cutting speed in the third direction.12. The process according to claim 1, wherein in the first direction, anamplitude a_(o) of vibration is used with a _(o)≧10 v _(c)/ω, wherein ωis the frequency in radians of the first vibrator and v_(c) is thecutting speed in the third direction.
 13. The process according to claim1, wherein a diamond blade is used.
 14. The process according to claim1, wherein the blade is moved relative to the probe in the seconddirection with a substantially constant cutting speed.